Rapamycin (U.S. Pat. Nos. 3,929,992 and 3,993,749) was reported as an antifungal antibiotic which was produced by Streptomyces hygroscopicus AY B-994 (ATCC 29253) (C. Vezina, A. Kudelski and S. N. Sehgal J. Antibiotics 28, 721-726, 1975). In recent years, it has been demonstrated that rapamycin shows potent immunosuppressive activity (Martel R. R. et al can. J. physiol. pharmcol. 55, 48-51, 1977). Rapamycin has been shown to be effective in inhibiting transplant rejection (U.S. patent application ser. no. 362, 544 filed Jun. 6, 1989). Rapamycin is widely used as an immunosuppressant in organ transplant recipients and has shown limited toxicities even in combination with other immunosupressants like cyclosporine or corticosteroids. The intracellular rapamycin receptor is a small protein termed FKBP12 (FK506-binding protein). The FKBP-rapamycin complex inhibits the function of a serine/threonine kinase, mTOR (mammalian target of rapamycin). In addition, rapamycin has in vitro and in vivo activity against a broad range of human tumor cell lines and considered to represent a promising new class of cytostatic anticancer agents.
The microbial process of rapamycin by aerobic fermentation of submersion culture of the new species Actinoplanes is described in WO9322446. Fermentation conditions like dissolved oxygen and air flow are not discussed. The titer of rapamycin produced by this organism is 405 mg/L only.
A method for producing rapamycin by solid state fermentation is disclosed in WO 2004/022767. The solid state fermentation process is difficult to adopt for larger volumes and scale up to commercial quantities. Determination of yield, titer value and biomass are difficult in this method. Substrates require pretreatment and it is very difficult to monitor process parameters like pH, dissolved oxygen and biomass concentrations, thus introducing batch to batch variations.
The present invention provides a titer value of 900 mg/L for rapamycin. A method for producing rapamycin by culturing the organism in an aqueous nutrient and novel fermentation media containing shikimic acid and allows the organism to produce the product in high yields. The process is well characterized with specifically identified process and engineering parameters to facilitate manufacture of rapamycin on a commercial scale.